Site-specific labelling of proteins using cyanine dye reporters

ABSTRACT

Disclosed are compounds of formula:  
                 
 
in which D is a dye selected from a cyanine dye or a derivative thereof; B is an affinity tag; F comprises a target bonding group selected from a carboxylic acid thioester group and a 1,2-aminothiol group; M is a group adapted for attaching to F; and L 1  and L 2  each independently comprise a group containing from 1 to 40 linked atoms selected from carbon atoms which may optionally include one or more groups selected from —NR′—, —O—, —CH═CH—, —CO—NH— and phenylenyl groups, where R′ is selected from hydrogen and C 1 -C 4  alkyl. The invention also relates to methods that afford direct attachment of the cyanine dye reporter group to either the N-terminus or C-terminus of a synthetic or recombinant peptide or protein, and their derivatives, in a site-specific manner, coupled with purification of the resultant labelled molecule.

The present invention relates to reagents and methods for site-specificlabelling of proteins using cyanine dyes as reporter molecules. Inparticular, the invention relates to new cyanine dye derivativescontaining thioester activated groups and groups reactive with targetmolecules containing or derivatised to contain a thioester reactivemoiety.

There is increasing interest in, and demand for, fluorescent reportersfor use in the labelling and detection of biomolecules. Cyanine andrelated dyes such as rigidised cyanine dyes and squaraines offer anumber of advantages over other fluorescent dye reagents and they arefinding widespread use as fluorescent labels in such diverse areas assequencing, microarrays, flow cytometry and proteomics. For example,U.S. Pat. No. 5,569,587 (Waggoner et al) discloses water soluble cyaninedye derivatives that possess reactive groups suitable for reaction withtarget molecules that contain, or are derivatised to contain, —OH, —NH₂,or —SH groups. The cyanine dyes are characterised by having very highextinction coefficients and favourable quantum yields. In addition,cyanine dyes possess good photostability and are not readilyphotobleached.

In many applications there is a need to form a permanent link, in theform of a covalent bond, between a fluorescent labelling dye and atarget molecule such as a protein. The chemistry of peptide and proteinlabelling is well documented and a wide range of labelling reagents arenow commercially available. For a review and examples of proteinlabelling using fluorescent labelling reagents, see “Non-RadioactiveLabelling, a Practical Introduction”, Garman, A. J. Academic Press,1997; “Handbook of Fluorescent Probes and Research Chemicals”, Haugland,R. P., Molecular Probes Inc., 1992).

Site-specific incorporation of a fluorescent label into a protein orpeptide may be of considerable benefit in certain biochemical andbiophysical studies, for example fluorescence resonance energy transfer,and protein structure and function studies. One method for thesite-specific attachment of a fluorescent label into a targetpolypeptide utilises the native chemical ligation reaction. According tothis procedure, an unprotected peptide fragment containing an N-terminalcysteine residue and a second unprotected peptide fragment containing anα-thioester group are chemoselectively ligated together at physiologicalpH, irrespective of their primary sequences, to generate an amide bondat the ligation site. For examples, see reviews by Cotton, G. J. andMuir T. W., Chem. Biol., (1999), 6, R247-260; Giriat, I., Muir, T. W.and Perler, F. B., Genetic Engineering, (2001), 23, 171-199; Muir, T.W., Syn. Lett., (2001), 6, 733-740.

Tolbert, T. J. and Wong, C-H. (Angew. Chem. Int. Ed., (2002), 41,2171-2174) describe the preparation of fluorescein and biotin thioesterderivatives and the reaction of these with N-terminalcysteine-containing recombinant proteins. Schuler, B. and Pannell, L. K.(Bioconjugate Chemistry, published on line, 18 Jul. 2002) reported thepreparation of a benzyl thioester of Cy5M and subsequent reaction with asynthetic polypeptide containing an N-terminal cysteine residue.

However, there are no reports describing thioester derivatives ofcyanine dyes in which the reporter is also linked covalently to anaffinity tag. Use of such a reagent in reactions involving site specificlabelling of proteins and peptides will be advantageous for subsequentseparation and purification of the fluorescent dye-labelled target. Thepresent invention therefore provides new cyanine dye reagents andmethods that afford direct attachment of the cyanine dye reporter toeither the N-terminus or C-terminus of a synthetic or recombinantpeptide or protein and their derivatives, in a site-specific manner,coupled with purification of the resultant labelled molecule.

According to one aspect of the present invention, there is provided acompound comprising a cyanine dye or derivative thereof containing, atleast one target bonding group selected from a carboxylic acid thioestergroup or a group suitable for covalent reaction with a thioester,characterised in that said compound includes an affinity tag covalentlybound thereto.

Suitably, the compound is of formula (I):

wherein:

-   D is a dye selected from a cyanine dye or a derivative thereof;-   B is an affinity tag;-   F comprises a target bonding group selected from a carboxylic acid    thioester group and a 1,2-aminothiol group;-   M is a group adapted for attaching to F; and-   L¹ and L² each independently comprise a group containing from 1-40    linked atoms selected from carbon atoms which may optionally include    one or more groups selected from —NR′—, —O—, —CH═CH—, —CO—NH— and    phenylenyl groups, where R′ is selected from hydrogen and C₁-C₄    alkyl.

Suitably, there are 2 to 30 atoms in each of L¹ and L², preferably, 6 to20 atoms.

Preferably, L¹ and L² are independently selected from the group:{(CHR′)_(p)-Q-(CHR′)_(r)}_(s)—where Q is selected from: —CHR′—, —NR′—, —O—, —CH═CH—, —Ar— and —CO—NH—;R′ is hydrogen or C₁-C₄ alkyl, p is 0-5, r is 1-5 and s is 1 or 2.

Particularly preferred Q is selected from: —CHR′—, —O— and —CO—NH—,where R′ is hereinbefore defined.

In one embodiment L² is a cleavable linker and may additionally includegroup P which may be suitably selected from a chemically-cleavablegroup, an enzyme-cleavable group, or a photochemically-cleavable group.Suitable chemically cleavable groups include carbamate esters andcarboxylate esters, which are both cleaved under basic conditions.Suitable enzyme cleavable groups may be selected from groups such asester, amide and phosphodiester groups. Such groups are substrates for,and are hydrolysed by hydrolases, such as proteases, esterases andphospho-diesterases. Suitable photocleavable groups P for use in thecompound of formula (I) may contain the 4,5-dialkoxy-2-nitrobenzylalcohol linker (Holmes, C. P., and Jones, D. G., J. Org. Chem., (1995),60, 2318-2319) or phenacyl linkers (Wang, S., J. Org. Chem., (1976), 41,3258-3261). These groups undergo efficient photoreaction upon 300 nmillumination, resulting in the rapid cleavage of the dye molecule ordye-labelled protein from the affinity tag.

Suitably, the group M may be any suitable functional group adapted forattaching the target bonding group F. Preferably, M is selected from:

wherein R′ is hereinbefore defined.

Suitable affinity tags may be selected from biotin, desthiobiotin andmetal chelating ligands such as his-tag and iminodiacetic acid,nitrilotriacetic acid and the like. Preferred affinity tags are selectedfrom biotin and desthiobiotin.

In one embodiment of the present invention, the target bonding group Fis a carboxylic acid thioester of formula:

wherein L′ is a bond or is a group containing from 1-30 linked atomsselected from carbon atoms and optionally one or more groups selectedfrom —NH—, —O— and —CO—NH—; and R″ is C₁-C₄ alkyl, C₆-C₁₀ aryl, orC₇-C₁₅ aralkyl, which may be optionally substituted with sulphonate; oris the group —(CH₂)₂CONH₂. In the case where L′ is a bond, the targetbonding group F is attached directly to group M.

In an alternative embodiment, the target bonding group F is a1,2-aminothiol group of formula:

wherein L′ is hereinbefore defined.

Thus, the present invention provides fluorescent labelling reagentscomprising a cyanine dye or derivative thereof, that are modified byincorporating a target bonding group and an affinity tag into themolecule. The target bonding group may be selected from a carboxylicacid thioester group or a 1,2-aminothiol group. Where the target bondinggroup is a thioester group, it is selectively reactive with a1,2-aminothiol group on a target molecule, suitably a protein orpeptide, or a derivative thereof. In the alternative, the cyanine dyemay contain a 1,2-aminothiol group for reaction with a thioester groupon the target. The incorporation of a reactive thioester or,alternatively, a 1,2-aminothiol functionality into the chemicalstructure of the reporter molecule enables the target molecule to bedirectly labelled in a convenient one step process. According to themethods of the invention, labelling of peptides and proteins issite-specific, irrespective of the composition of the primary sequence.By generating the target primary sequence with either an N-terminalcysteine or a thioester functionality, site-specific labelling can beachieved directly, by incubating the target with the appropriatederivative of the cyanine dye, suitably, the thioester and1,2-aminothiol derivatives respectively. Furthermore, inclusion of anaffinity tag in the labelling reagent allows subsequent purification ofthe fluorescent dye labelled protein or peptide.

Suitably, the cyanine dye or cyanine dye derivative may be selected fromcyanine dyes, rigidised cyanine dyes and squaraine dyes, provided thatthe dye incorporates at least one carboxylic acid thioester group, or agroup suitable for covalent reaction with a thioester. Table 1 showssome examples of cyanine dyes, having particular excitation (Abs) andemission (Em) characteristics. TABLE 1 Dye Fluorescence Colour Abs (nm)Em (nm) Cy2 Green 489 506 Cy3 Orange 550 570 Cy3.5 Scarlet 581 596 Cy5Far red 649 670 Cy5.5 Near-IR 675 694 Cy7 Near-IR 743 767

In one embodiment according to the first aspect, the compound has theformula (II):

wherein:

-   groups R³ and R⁴ are attached to the Z′ ring structure and groups R⁵    and R⁶ are attached to the Z² ring structure;-   n is an integer from 1 to 3;-   Z¹ and Z² independently represent the atoms necessary to complete    one ring or two fused ring aromabc or heteroaromatic systems, each    ring having five or six atoms selected from carbon atoms and    optionally no more than two atoms selected from oxygen, nitrogen and    sulphur;-   X and Y are the same or different and are selected from: >CR⁸R⁹,    oxygen, sulphur, —CH═CH—, >N—W wherein N is nitrogen and W is    selected from hydrogen and the group R¹⁰;-   at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹ and R¹⁰ is the    group:    where B, F, M, L¹ and L² are hereinbefore defined; groups R⁷ are    independently selected from hydrogen and C₁-C₄ alkyl which may be    unsubstituted or substituted with aryl, or two or more of R⁷    together with the group:    form a hydrocarbon ring system substituted with R⁷ and which may    optionally contain a heteroatom selected from —O—, —S— or >NR⁷,    wherein R⁷ and n are hereinbefore defined;-   remaining groups R³, R⁴, R⁵ and R⁶ are independently selected from    the group consisting of hydrogen, halogen, amide, cyano, nitro,    mono- or di-C₁-C₆ alkyl-substituted amino, carbonyl, carboxyl, C₁-C₆    alkyl, C₁-C₆ alkoxy, aryl, heteroaryl, aralkyl and the group    —(CH₂)_(m)—Y where Y is selected from sulphonate, sulphate,    phosphonate, phosphate and quaternary ammonium and m is zero or an    integer from 1 to 6;-   remaining groups R⁸, R⁹ and R¹⁰ are independently C₁-C₆ alkyl; and    remaining groups R¹ and R² are independently selected from hydrogen,    C₁-C₁₀ alkyl, the group —(CH₂)_(m)—Y wherein Y and m are    hereinbefore defined, and benzyl which may be unsubstituted or    substituted by up to two nitro groups.

In a second embodiment according to the first aspect, the compound hasthe formula (III):

wherein

-   groups R¹², R¹³, R¹⁴ and R¹⁵ are attached to the rings containing X    and Y or, optionally are attached to atoms of the Z¹ and Z² ring    structures;-   Z¹ and Z² independently represent the atoms necessary to complete    one ring or two fused ring aromatic or heteroaromatic systems, each    ring having five or six atoms selected from carbon atoms and    optionally no more than two atoms selected from oxygen, nitrogen and    sulphur;-   X and Y are the same or different and are selected from: >CR⁸R⁹,    oxygen, sulphur, —CH═CH—, >N—W wherein N is nitrogen and W is    selected from hydrogen and the group R¹⁰;-   A is selected from O and NR¹⁶ where R¹⁶ is the substituted amino    radical:    at least one of groups R⁸, R⁹, R¹⁰ R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁷ and    R¹⁸ is the group:    where B, F. M, L¹ and L² are hereinbefore defined;-   remaining groups R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ are independently    selected from the group consisting of hydrogen, halogen, amide,    cyano, nitro, amino, mono- or di-C₁-C₆ alkyl-substituted amino,    carbonyl, carboxyl, C₁-C₆ alkyl, C₁-C₆ alkoxy, aryl, heteroaryl,    aralkyl and the group —(CH₂)_(m)—Y where Y is selected from    sulphonate, sulphate, phosphonate, phosphate and quaternary ammonium    and m is zero or an integer from 1 to 6;-   remaining groups R⁸, R⁹ and R¹⁰ are independently C₁-C₆ alkyl;    remaining group R¹⁷ is selected from hydrogen, C₁-C₄ alkyl and aryl;    and remaining group R¹⁸ is selected from C₁-C₆ alkyl, aryl,    heteroaryl, an acyl radical having from 2-7 carbon atoms, and a    thiocarbamoyl radical.

Suitably, in the compounds according to formula (II) and (III), Z¹ andZ² may be selected independently from the group consisting of phenyl,pyridinyl, naphthyl, anthranyl, indenyl, fluorenyl, quinolinyl, indolyl,benzothiophenyl, benzofuranyl and benzimidazolyl moieties. Additionalone, or two fused ring systems will be readily apparent to the skilledperson. Preferably, Z¹ and Z² are selected from the group consisting ofphenyl, pyridinyl, naphthyl, quinolinyl and indolyl moieties.Particularly preferred Z¹ and Z² are phenyl and naphthyl moieties.

Suitably, at least one of the groups R of the compounds of formula (II)and (III) is a water solubilising group for conferring a hydrophiliccharacteristic to the compound. Solubilising groups, for example,sulphonate, sulphonic acid and quaternary ammonium, may be attacheddirectly to the aromatic ring structures Z¹ and/or Z² of the compoundsof formula (II) and (III). Alternatively, solubilising groups may beattached by means of a C₁ to C₆ alkyl linker chain to said aromatic ringstructures and may be selected from the group —(CH₂)_(m)—Y where Y isselected from sulphonate, sulphate, phosphonate, phosphate, quaternaryammonium and carboxyl; and m is hereinbefore defined. Alternativesolubilising groups may be carbohydrate residues, for example,monosaccharides, or polyethylene glycol derivatives. Examples of watersolubilising constituents include C₁-C₆ alkyl sulphonates, such as—(CH₂)₃—SO₃— and CH₂)₄—SO₃—. However, one or more sulphonate orsulphonic acid groups attached directly to the aromatic ring structuresof a dye of formula (II) or (III) are particularly preferred. Watersolubility may be advantageous when labelling proteins.

In one embodiment the compound of formula (I) is a fluorescent reportermolecule. In this embodiment, none of the substituent groups R in thecompounds of formula (II) and (III) contains a nitro group.

In another embodiment, the compound of formula (I) is non-fluorescent orsubstantially non-fluorescent dye wherein at least one of the groups Rattached to the aromatic ring structures of the compounds of formula(II) and (III) comprises at least one nitro group. In this embodiment,suitably, the at least one nitro group may be attached directly to theZ¹ and/or Z² ring structures. In the alternative, a mono- ordi-nitro-substituted benzyl group may be attached to the Z¹ and/or Z²ring structures which optionally may be further substituted with one ormore nitro groups. The non-fluorescent or substantially non-fluorescentcyanine dye or cyanine dye derivatives according to the invention may beused to label one component of a fluorescent donor/acceptor pair inassays involving the detection of binding and/or cleavage events inreactions involving biological molecules, as described in EP 1086179 B1(Amersham Biosciences UK Limited).

In the embodiments according to the first aspect:

-   i) Aryl is an aromatic substituent containing one or two fused    aromatic rings containing 6 to 10 carbon atoms, for example phenyl    or naphthyl, the aryl being optionally and independently substituted    by one or more substituents, for example halogen, straight or    branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl    and alkoxy for example methoxy, ethoxy, propoxy and n-butoxy;-   ii) Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ring    system containing at least one and no more than 3 heteroatoms which    may be selected from N, O, and S and is optionally and independently    substituted by one or more substituents, for example halogen,    straight or branched chain alkyl groups containing 1 to 10 carbon    atoms, aralkyl and alkoxy for example methoxy, ethoxy, propoxy and    n-butoxy;-   iii) Aralkyl is a C₁-C₆ alkyl group substituted by an aryl or    heteroaryl group;    -   iv) Halogen and halo groups are selected from fluorine,        chlorine, bromine and iodine.

By virtue of the target bonding group F, the compounds according to thepresent invention are useful for covalently labelling target biologicalmaterials in a site specific manner for applications in biologicaldetection systems. Suitable target materials include proteins,post-translationally modified proteins, peptides, antibodies, antigens,and protein-nucleic acids (PNAs). The reporter moiety may also beconjugated to species which can direct the path of the reporter withinor aid entry to or exit from cells (live or dead); such as for example,long alkyl residues to allow permeation of lipophilic membranes, orintercalating species to localise a reporter in a nucleus or othercellular enclave containing double-stranded DNA.

In a second aspect, there is provided a method for labelling a proteinof interest wherein said protein contains or is derivatised to containan N-terminal cysteine, the method comprising:

-   i) adding to a liquid containing said protein a compound of formula    (I):    wherein:-   D is a dye selected from a cyanine dye or a derivative thereof;-   B is an affinity tag;-   F comprises a target bonding group selected from a carboxylic acid    thioester group and a 1,2-aminothiol group;-   M is a group adapted for attaching to F; and-   L¹ and L² each independently comprise a group containing from 1-40    linked atoms selected from carbon atoms which may optionally include    one or more groups selected from —NR′—, —O—, —CH═CH—, —CO—NH— and    phenylenyl groups, where R′ is selected from hydrogen and C₁-C₄    alkyl; and-   ii) incubating said compound with said protein under conditions    suitable for labelling said protein.

Suitably, there are 2 to 30 atoms in each of L: and L², preferably, 6 to20 atoms.

Preferably, L¹ and L² are independently selected from the group:—{(CHR′)_(p)-Q-(CHR′)_(r)}_(s)—where Q is selected from: —CHR′—, —NR′—, —O—, —CH═CH—, —Ar— and —CO—NH—;R′ is hydrogen or C₁-C₄ alkyl, p is 0-5, r is 1-5 and s is 1 or 2.

Particularly preferred Q is selected from: —CHR′—, —O— and —CO—NH—,where R′ is hereinbefore defined.

Preferred compounds of formula (I) for use in labelling a target proteinare those having formula (II) or (III) as hereinbefore defined.

Covalent labelling using compounds of the present invention may beaccomplished with a target having at least one carboxylic acid thioestergroup or 1,2-aminothiol group as hereinbefore defined. The target may beincubated with an amount of a compound of the present invention havingat least one group F as hereinbefore defined that can covalently bindwith the complementary group of the target material. The target materialand the compound of the present invention are incubated under conditionsand for a period of time sufficient to permit the target material tocovalently bond to the compound of the present invention. Thus, forexample, the thioester group F may be reacted and form a covalent bondwith any of the above target materials that contains, or has beenderivatised to contain, a 1,2-amino thiol group. These methods and theproducts resulting from them, for example, reporter-labelledbiomolecules are envisaged as further aspects of the invention.

Suitably, the protein of interest may be selected from the groupconsisting of antibody, antigen, protein, peptide, microbial materials,cells and cell membranes.

In a particular embodiment according to the second aspect, there isprovided a method of separating and/or purifying the dye-labelledprotein of interest by affinity chromatography utilising the affinity ofthe affinity tag moiety for an immobilised ligand (or specific bindingpartner) attached to a support material. Affinity chromatographyprovides a quick and convenient method to enable the separation oflabelled and unlabelled protein molecules under physiologicalconditions. Proteins labelled with an affinity tag can be selectivelybound to an affinity column and any unreacted protein removed by washingthe column. Suitable specific binding moieties include avidin orstreptavidin (for a biotin tag); immobilised metal ions, for example,Cu(II), Ni(II), Fe(II) and Fe(III) (for His-tag or iminodiacetic acid).Methods for affinity purification of proteins will be well known to theskilled person, see for example Ostrove, S, Methods in Enzymology,(1990), Vol 182, page 357.

In a typical labelling procedure, a target peptide or protein containingan N-terminal cysteine residue is agitated with an excess of a cyaninedye thioester derivative, e.g. Cy5-MESNA (Cy5-mercaptoethanesulphonicacid ester), in phosphate buffer (typically 200 mM NaCl, 200 mM sodiumphosphate) at ˜pH 7.3-7.4 containing ˜1.5% MESNA. The concentration ofthe target polypeptide in the labelling reaction is generally between100 μM to 10 mM, whilst the Cy5-MESNA is generally present in excess,for example 1.5 to 3-fold molar excess. When the target polypeptideconcentration is relatively low the concentration of Cy5-MESNA isusually maintained at or above 1 mM. Generally, for labelling smallpeptides a solution of Cy5-MESNA and MESNA cofactor is directly added tothe lyophilised target.

Typically, for site specific labelling of proteins and largepolypeptides using the reagents of the present invention, the target isfirst exchanged into an appropriate buffer, which is known not to affectthe labelling reaction. An equal volume of a solution of Cy5-MESNA andMESNA thiol cofactor in ligation buffer is then added to the protein togive the desired final concentration of the reactants. The reactionmixture is agitated overnight at room temperature. The reaction time maybe lowered to less than one hour for high reactant concentrations or, ifthe stability of the target polypeptide is an issue, the labellingreaction may be performed efficiently at 4° C. On completion of thelabelling reaction, dithiothreitol (DTT) is added to a finalconcentration of −50 mM and the desired material isolated by affinitychromatography.

Various different denaturants, organic solvents and detergents may beadded to the reaction buffer when performing native chemical ligationand expressed protein ligation reactions, to aid the ligation of thepeptide fragments and/or stabilise the reactants or products. Suchreagents may be utilised in the labelling reaction to increase productyield if necessary. Examples include, but are not limited to guanidiniumchloride, urea, dimethylformamide, dimethylsulphoxide, acetonitrile,triton X-100, octyl glucoside, 1,6-hexanediol and glycerol.

The ligation reaction using the derivatised cyanine dye according to thepresent invention may be optimally performed at between pH 7.0 and pH8.0 and at temperatures varying between 4° C. and 37° C. It is envisagedthat such a range of conditions are compatible to the site-specificlabelling reaction described herein.

The advantage of the present method is that it enables the introductionof an extrinsic label into a proteinacious substrate in a regioselectiveand specific manner, thus minimising any detrimental effects thatlabelling may have on the biological function of the protein. Theimportance of controlling stoichiometry of labelling is important wheredye overload may interfere with biological activity. In addition, ifthis controlled labelling stoichiometry is directed towards a singleterminal site, rather than towards an internal site, this may have thebenefit of further maintaining the biological viability of the labelledspecies.

The invention is further illustrated by reference to the followingexamples and figure in which:

FIG. 1 illustrates the products from the labelling reaction of anN-terminal cysteine derivative of the Grb2SH2 domain with the thioesterderivative, α-D-desthiobiotin-ε-Cy5-L-lysine-MESNA according to Examples3 and 4.

EXPERIMENTAL

1.2-[(1E,3E,5E)-5-(3,3-Dimethyl-1-{6-oxo-6-[(2-sulphoethyl)thio]hexyl}-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-1-ethyl-3.3-dimethyl-5-sulfo-3H-indolium

To Cy™5 mono acid (47 mg, 0.062 mmol) in a solution of7-azobenzotriazolyoxytris(pyrrolidino)phosphonium hexafluorophosphate(PyAOP, 66 mg, 0.127 mmol) in anhydrous dimethylformamide (DMF, 1 ml)was added anhydrous di-isopropylethylamine (DIEA)(30 μl, 0.1724 mmol)and mixed for 5 minutes. The activated dye solution was then added to astirred solution of 2-mercaptoethanesulphonic acid, sodium salt (MESNA,40 mg, 0.243 mmol) in DMF (2 mls) and DIEA (30 μl, 0.1724 mmol) under adry nitrogen atmosphere. To this mixture was added as a solid, dried 4Amolecular sieves (˜1 g, <5 micron, activated powder). The mixture wasstirred under a dry nitrogen atmosphere, at room temperature, in thedark overnight. Thin layer chromatography analysis (reverse phase C18plates, eluents water/acetonitrile (70:30, containing 0.1% TFA)indicated a major component, Rf_(thioester)=0.25) with no trace ofstarting material (Rf_(acid)=0.12).

The molecular sieves were removed by filtration and filtrate was addeddropwise into an excess of ethyl acetate, the blue solid was filteredoff and was purified by reverse phase-high performance liquidchromatography (RP-HPLC); [Phenomenex Prodigy C18 column; 15% B-30% Bover 30 mins @ 20 ml/min; eluent A=0.1% TFA/water, eluent B=0.1%TFA.MeCN, UV detection at 650 nm]. The product was isolated as a darkblue/purple solid (40 mg, 0.0513 mmol, 83% yield).

Accurate mono-isotopic mass: C₃₅H₄₅O₁₀N₂S₄ requires 781. Found MaldiTof, LC-MS found mass: M+781.25. δ H (300 MHz, d6-DMSO): 8.37 (t, 1H),8.36 (t, 1H), 7.83 (d, 1H), 7.82 (d, 1H), 7.67(dd, 1H), 7.64 (dd, 1H),7.36 (d, 1H), 7.33 (d, 1H), 6.61 (t, 1H), 6.38 (d, 1H), 6.28 (d, 1H),4.15 (m, 2H), 4.08 (t, 2H), 3.06 (m, 2H), 2.63 (m, 2H), 2.56 (t, 2H),1.64 (m, 2H), 1.28(t, 3H, 7.1), 1.40 (m, 2H). λ_(max) (abs>=647 nm.(ε(H₂O)=230,000 M⁻¹ cm⁻¹).

2. Determination of Specificity of Labelling using2-[(1E,3E,5E)-5-(3,3-dimethyl-1-{6-oxo-6-[(2-sulphoethyl)thio]hexyl}-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium

2.1 Preparation of Cv5-Cys-Gly-Leu-Asp-Lys-Arg-Gly-Cys-Gly-NH₂

i) Synthesis ofH-Cys(Trt)-Gly-Leu-Asp(OtBu)-Lys(Boc)Arg(Pmc)-Gly-Cys(Trt)-Gly-rinkamide resin

H-Cys(Trt)-Gly-Leu-Asp(OtBu)Lys(Boc)-Arg(PmcyGly-Cys(Trt)-Gly-rink amideresin was synthesised using a commercially available Applied BiosystemsModel 433A automated peptide synthesiser using FastMoc™ chemistry,following the instrument manufacturer's recommended proceduresthroughout. The peptide was synthesised on a 0.25 millimolar scaleemploying O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) as the activating agent.ii) H-Cys-Gly-Leu-Asp-Lys-Arg-Gly-Cys-Gly-NH₂

H-Cys(Trt)-Gly-Leu-Asp(OtBu)-Lys(Boc)-Arg(Pmc)-Gly-Cys(Trt)Gly-rinkamide resin (100 mg, theoretical loading 0.36 mmol/g) was deprotectedand cleaved from solid phase in 95% trifluoroacebc acid (TFA)/2.5%triisopropylsilane (TIS)/2.5% water (3 mis) at room temperature for 2hours. The crude product was precipitated into a 10 fold excess of colddiethyl ether, centrifuged at 2500 rpm for 5 minutes and the etherdecanted off. The crude peptide was washed twice more with ether and waspurified by reverse phase-high performance liquid chromatography(RP-HPLC) [Phenomenex Jupiter C18 column, eluent A: 0.1% TFA/water,eluent B: 0.1% TFA/acetonitrile, gradient: 0-73% B over 30 mins @1ml/min, detection at 214 nm]. The product was isolated and lyophilisedto afford a colourless fluffy solid (21 mg by weight, 60%).Mono-isotopic mass: 906.4. Found mass (LC-MS): MH+@ 907.3;

-   -   M+Na @ 929.6; >95% pure as judged by RP-HPLC @ 214 nm        (Phenomenex Jupiter C18 column, eluent A: 0.1% TFA/water, eluent        B: 0.1% TFA/acetontrile, 5-50% B over 25 mins @ 1 ml Imin, UV        detection at 650 nm).        iii) Cv5-Cys-Gly-Leu-Asp-Lvs-Arg-Gly-Cys-Gly-NH₂

To solid H-Cys-Gly-Leu-Asp-Lys-Arg-Gly-Cys-Gly-NH2 (3.0 mg by weight,0.0033 mmol) was added a solution of Cy5-MESNA (3.5 mg, 0.0045 mmol) in200 mM phosphate buffer, 200 mM NaCl pH 7.2 containing 1.5%2-mercaptoethanesulphonic acid, sodium salt (400 μl). The reactionmixture was stirred on rollers for 30 minutes at room temperature indarkness. During incubation, a blue precipitate formed, whichre-dissolved on addition of acetonitrile (40 μl).

500 mM DTT (200 μl) in 200 mM phosphate buffer; 200 mM NaCl pH 7.2 (0.5mis, 0.0025 mmol) was then added to the reaction mixture, with completemixing and was stirred for a further 30 minutes at room temperature inthe dark. The crude reaction mixture was then purified by RP-HPLC[Phenomenex Jupiter C18 column, eluent A: 0.1% TFA/water, eluent B: 0.1%TFA/acetontrile, gradient; 20-35% B over 30 mins at 4 ml/min, detectionat 650 nm and 214 nm]. The product was isolated and lyophilised as ablue fluffy solid (1.6 mg by UVNIS at 650 nm; 50% Yield; 98% pure asjudged by RP-HPLC at 650 nm. Mono-isotopic mass C₆₇H₁₀₁N₁₆O₁₈S₄ requires1545.636. Found (LC-MS) M+1545.7.

2.2 Characterisation of Labelled Peptide

i) Ellman's Test on Cy5-Cys-Gly-Leu-Asp-Lys-Arg-Gly-Cys-Gly-NH₂

A sample of Cy5-Cys-Gly-Leu-Asp-Lys-Arg-Gly-Cys-Gly-NH₂ was dissolved in100 mM sodium phosphate buffer; 1 mM EDTA pH 7.27 (stock buffer) toafford a 0.3 μM peptide stock by UVNIS at 650 nm.

0.3 μM peptide stock (40 μl) and 10 mM 5,5′-dithiobis(2-nitrobenzoicacid (DTNB) in 100 mM sodium phosphate buffer; 1 mM EDTA pH 7.27 (50 μl)were mixed together in stock buffer (910 μl) to afford a green solution.The absorbance at 412 nm (due to generation of TNB²⁻) was recordedagainst a DTNB blank [10 mM DTNB stock (50 μl) in stock buffer (950 μl].Using the known molar absorption coefficient of TNB²⁻ (14150M⁻¹ cm⁻¹),the thiol concentration was determined as 655 μM, approximately twicethe peptide concentration, confirming two free thiol groups. [SH]=[A412nm (sample)-A412 nm(reference)/ε (TNB²⁻)

ii) Enzyme Digestion of Cy5-Cys-Gly-Leu-Asp-Lys-Arg-Gly-Cys-Gly-NH₂

To a solution of Cy5-Cys-Gly-Leu-Asp-Lys-Arg-Gly-Cys-Gly-NH₂ (180 μg byUV/VIS at 650 nm) in TRIS buffer pH 8.0 (100 μl) containing 10%acetonitrile was added Asp-N (2 μg) in TRIS buffer pH 8.0 (70 μl). Thereaction mixture was stirred at room temperature in the dark for 4hours. The reaction mixture was treated with 250 mM Tris(2-carboxyethyl)phosphine, HCL (TCEP) in TRIS buffer pH 8.0 (55 μl) for30 minutes. The reduced reaction mixture was then diluted 1:5 with 0.1%TFA in water and purified by reverse phase HPLC [Phenomenex Jupiter C18,eluent A: 0.1% TFA/water, eluent B: 0.1% TFA/acetonitrile, 5-50% B over30 mins @ 1 ml/min, UV at 214 nm, 650 nm]. The two components of thereaction mixture were identified as: Cy5-Cys-Gly-Leu-OH, mono-isotopicmass: C₄₄H₆₀N₅O₁₁S₃ requires 930.3451. Found mass (MALDI Tof): M+930.0and H-Asp-Lys-Arg-Gly-Cys-Gly-NH₂, monoisotopic mass: C₂₃H₄₃N₁₁O₈Srequires 633.3016, found mass (MALDI-Tof): M+633.0.3. Preparation of α-D-Desthiobiotin-ε-Cy5-L-lysine-MESNA[N⁶-(6-{(2Z)-2-[(2E,4E)-5-(1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium-2-yl)penta-2,4-dienylidenel-3.3-dimethyl-5-sulfo-2,3-dihydro-1H-indol-1-yl}hexanoyl)-N²-(6-{(6-(5-methyl-2-oxoimidazolidin-4-yl)hexanoyl]amino}hexanoyl)lysylthioethane-2-sulfonic acid]

3.1 Preparation of α-Fmoc-c-Cy5-L-lysine-OH[2-[(1E,3E)-5-(1-(6-[(5-carboxy-5-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}pentyl)amino]-6-oxohexy}-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)-1,3-pentadienyl]-1-ethyl-3,3-dimethyl-5-sulfo-3H-indoliumsalt]

Cy5 mono free acid potassium salt (Amersham Pharmacia Biotech Ltd) (450mg, 0.65 mmol) and DIEA (720 μl) were dissolved in anhydrousdimethylsulphoxide (18 ml). To this was addedO-(N-succinimidyl)-N,N,N′,N′-bis(tetramethylene)-uroniumhexafluorophosphate (666 mg, 1.6 mmol) and the reaction mixture stirredat room temperature for 1 hr after which time negligible startingmaterial remained by TLC (RPC₁₈, 1:1 methanol:water). The reactionmixture was slowly poured into diethyl ether to precipitate the product;Cy5 mono NHS ester, which was filtered off, washed with ethyl acetateand dried in vacuo. The product was re-dissolved in anhydrousdimethylsulphoxide (18 ml) and DIEA (720 μl) added. Fmoc-lysine-OH (360mg, 0.98 mmol) was suspended in a mixture of phosphate buffer (pH 7.4)(9 ml) and dimethylsulphoxide (9 ml). The suspension was slowly added tothe solution of Cy5 NHS ester. The reaction mixture was stirred at roomtemperature for 12 hours. TLC (RPC₁₈, 2:3 methanol:water) showed thedisappearance of starting material and the formation of a new productspot.

The product was purified by HPLC (Dynamax C18 column (50×4.14 cm); flowrate 25 ml/min; gradient of 20 to 80% B over 80 mins (eluent A=0.1% TFAin water and eluent B=0.1% TFA in acetonitrile); detection at 650 nm.The fractions containing the desired product were pooled and most of thesolvent removed under reduced pressure, the residue was freeze dried.The product; α-Fmoc-c-Cy5-L-lysine-OH was obtained as a fluffy cyansolid (487 mg, 74%). MS (MALDI TOF) found 1008(M⁺); [theoretical(C₅₄H₆₄N₄O₁₁S₂) 1009]. ¹H NMR (200 MHz D₆DMSO) 1.27(t, 3H, CH₃), 1.35(m, 4H, CH₂, CH₂), 1.55 (m's, 4H, CH₂, CH₂), 1.7 (s, 12H, (CH₃)₂), 1.78(m, 2H, CH₂), 2.05 (t, 2H, CH₂), 3.0, (m, 2H, CH₂), 3.92 (m, 1H CH aminoacid), 4.11 (m, 4H, N—CH₂, N⁺CH₂), 4.27 (m 3H O—CH₂, CH fluorenyl), 6.3(d, 2H, α, α′ methine), 6.59, (t, 1H, γ methine) 7.28-7.48 (m's, 6H,Fmoc and indole Ar), 7.65 (d, 2H, fluorenyl Ar), 7.73 (d, 2H, fluorenylAr), 7.85 (s, 2H, indole Ar), 7.9 (d, 2H, indole Ar), 8.38 (t, 2H, β,β′methine).

3.2 Preparation of E-Cy5-L-lysine-OH[N⁶-(6-{(2E)-2-[(2E,4E)-5-(1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium-2-yl)penta-2,4-dienylidene]-3,3-dimethyl-5-sulfo-2,3-dihydro-1H-indol-1-yl}hexanoyl)lysine]

α-Fmoc-c-Cy5-L-lysine-OH (100 mg, 0.1 mmol) was deprotected in a mixtureof 20% piperidine in NMP (2 ml). TLC (RP C18; 1:1 MeOH:water) showed theformation of a new product spot, rf=0.92 as compared to that of thestarting material, rf=0.46. The piperidine was removed under reducedpressure and the dye precipitated by pouring the reaction mixture intodiethyl ether. The product was filtered off and washed withdichloromethane and then ethyl acetate to remove the yellow Fmoc derivedby-product. The product was dissolved in water, filtered and thenpurified by HPLC; [Vydac Protein and Peptide C18 column; 0-50% B over 45mins at 10 ml/min; eluent A=0.1% TFA/water, eluent B=0.1% TFA/MeCN,detection at 215 nm). Fractions containing the desired product werecombined and the solvents removed under reduced pressure to leave a blueresidue. The residue was triturated with ethyl acetate and the resultantsolid dried under vacuum at 40° C. The product; c-Cy5-L-lysine-OH wasobtained as a dark blue solid (43 mg, 48%). Analytical HPLC AKTAanalysis; Phenomenex C18 column; 0-50% B over 30 mins at mli/min; eluentA=0.1% TFA/water, eluent B=0.1% TFA/MeCN, detection at 650 nm; rt=20.22mins. MS (MALDI TOF) found 785 (M⁺); [theoretical (C₃₉H₅₃N₄O₉S₂) 785].¹H NMR (300 MHz D₆DMSO) 1.26 (t, 3H, CH₃), 1.52 (m, 4H, CH₂, CH₂), 1.62(m, 4H, CH₂, CH₂), 1.69 (S, 12H, (CH₃)₂), 2.02 (m, 2H, CH₂), 2.92 (m,2H, CH₂), 3.85 (m, 1H, CH amino acid), 4.10 (m, NCH₂, N⁺CH₂), 6.29 (d,1H a methine), 6.34 (d, 1H, α′ methine), 6.58 (t, 1H, γ methine), 7.32(m, 2H, indole Ar), 7.82 (d, 2H, indole Ar), 8.04 (m, 3H, NH₃ ⁺), 8.37(t, 2H, β,β′ methine).

3.3 Preparation of D-Desthiobiotinamidocaproic Acid

D-Desthiobiotin (250 mg, 1.17 mmol) was dissoved in anhydrousdimethylsulphoxide (2 ml). To this solution was added PyAOP (610 mg,1.17 mmol) and DIEA (200 μl, 1.15 mmol). The mixture was stirred undernitrogen at RT for 3 hrs before adding 6-aminocaproic acid (153 mg, 1.17mmol) and a further amount of DIEA (200 μl, 1.15 mmol). The reactionmixture was stirred for a further 4 hrs. TLC (RP C18; 1:2 MeOH:water;detection by cinnamaldehyde staining) showed the formation of a newproduct spot, rf=0.63 as compared to the starting material, rf=0.76).The reaction mixture was poured into excess diethyl ether to give abrown oil. The oil was triturated with ethyl acetate until an off-whitesolid was obtained. The product was filtered off and purified by HPLC[Vydac Protein and Peptide C18 column; 0-50% B over 30 mins at 10ml/min; eluent A=0.1% TFA/water, eluent B=0.1% TFA/MeCN, detection at215 nm). Fractions containing the desired product were pooled and thesolvents removed under reduced pressure. The residue was triturated withethyl acetate to give a white solid. The product was filtered off anddried under reduced pressure at 40° C. The product;D-desthiobiotinamidocaproic acid, was obtained as a white solid (48 mg,12.5%). MS (MALDI TOF) found 327 (M+); [theoretical (C₁₆H₂₉N₃O₄) 327].¹H NMR (300 MHz D₆DMSO) 0.96 (d, 3H, CH₃), 1.25 (m, 6H, CH₂, CH₂, CH₂),1.34 (m, 4H, CH₂, CH₂), 1.48 (m, 4H, CH₂, CH₂), 2.03 (m, 2H, C(O)CH₂),2.60 (m, 2H, C(O)CH₂), 3.01 (m, 2H, NHCH₂), 3.47 (m, 1H, CH), 3.60 (m,1H, CH), 6.11 (s, 1H, NH), 6.29 (s, 1H, NH), 7.71 (s, 1H, NH).

3.4 Preparation of D-Desthiobiotinamidocaproate N-hydroxy SuccinimidylEster

D-Desthiobiotinamidocaproic acid (48 mg, 0.147 mmol) was dissolved inDMF (1 ml) and N,N,N′,N′-bis(tetramethylene)-O-(N-succinimidyl)uroniumhexafluorophosphate (HSPyU) (90 mg, 0.21 mmol) and DIEA (40 μl, 0.23mmol) were added. The reaction mixture was stirred under nitrogen at RTfor 6 hrs, TLC (RP C18; 1:2 MeOH:water; materials detected bycinnamaldehyde staining) showed the formation of a new product at thebase line as compared to the starting material, rf=0.68. The reactionmixture was poured into diethyl ether to give a brown gum. Thesupernatant was decanted off and the gum again treated with diethylether. No solid formed. The gum was dried under reduced pressure and theproduct, D-desthiobiotinamidocaproate N-hydroxy succinimidyl ester wasused directly in the next dye coupling reaction, assuming a theoreticalyield of 62 mg.

3.5 Preparation of α-D-Desthiobiotin-ε-Cy5-L-lysine-OH[N⁶-(6-(2Z)-2-[(2E,4E)-5-(1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium-2-yl)penta-2,4-dienylidenel-3.3-dimethyl-5-sulfo-2,3-dihydro-1H-indol-1-yl]hexanoyl)-N²-(6-{[6-(5-methyl-2-oxoimidazolidin-4-yl)hexanoyl]amino}hexanoyl)lysine]

ε-Cy5-L-lysine-OH (43 mg, 0.048 mmol), D-desthiobiotinamidocaproateN-hydroxy succinimidyl ester (62 mg, 0.146 mmol) and DIEA (80 μl, 0.45mmol) were stirred together in DMF (2 ml) for 3 hrs. TLC (RP C18; 1:1MeOH:water) showed the formation of a new product spot, rf=0.79, justunder that of the starting material. The product was precipitated intodiethyl ether (200 ml) and then filtered off. The material was purifiedin multiple runs by HPLC [Vydac Protein and Peptide C18 column; eluentA=0.1% TFA/water, eluent B=0.1% TFA/MeCN, various gradients, detectionat 215 nm) until the material was seen to be pure by ¹H NMR. AnalyticalHPLC AKTA analysis; Phenomenex C18 column; 0-50% B over 30 mins at 1ml/min; eluent A=0.1% TFA/water, eluent B=0.1% TFA/MeCN, detection at650 nm; rt=22.04 mins. MS (MALDI TOF) found 1094 (M⁺); [theoretical(C₅₅H₈₀N₇O₁₂S₂) 1094].

3.6 Preparation of α-D-Desthiobiotin-c-Cy5-L-lysine-MESNA[N⁶-(6-{(2Z)-2-[(2E,4E)-5-(1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium-2-yl)penta-2,4-dienylidene]-3,3-dimethyl-5-sulfo-2,3-dihydro-1H-indol-1-yl}hexanoyl)-N²-(6-{[6-(5-methyl-2-oxoimidazolidin-4-yl)hexanoyl]amino}hexanoyl)lysylthiopropane-3-sulfonic acid]

α-D-Desthiobiotin-F-Cy5-L-lysine-OH (10 mg, 8.8 μmol) was dissolved inanhydrous dimethylsulphoxide (2 ml), PyAOP (10 mg, 19.2 μmol), MESNA (5mg, 0.30 mmol) and DIEA (10 μl, 0.06 mmol) were added and the reactionmixture was stirred under nitrogen for 4 hrs. The reaction mixture waspurified by RP-HPLC; [Vydac Protein and Peptide C18 column; 15-40% Bover 45 mins at 10 ml/min; eluent A=0.1% TFA/water, eluent B=0.1%TFA/MeCN, detection at 215 nm). The product containing fractions werecombined and the majority of solvent removed under reduced pressure, theresidue was freeze dried. The product was obtained as a fluffy bluesolid (4 mg, 37%). TLC; RP C18; 1:1 water:acetonitrile) rf=0.76.Analytical HPLC AKTA analysis; Phenomenex C18 column; 0-50% B over 30mins at 1 ml/min; eluent A=0.1% TFA/water, eluent B=0.1% TFA/MeCN,detection at 650 nm; rt=21.04 mins. λmax=648 nm (PBS buffer). MS (MALDITOF) found 1219 (MH⁺); [theoretical (C₅₇H₈₄N₇O₁₄S₄) 1218].

4. Protein Labelling and Affinity Purification

4.1 Labelling of N Terminal Cysteine Grb2SH2 withα-D-desthiobiotin-ε-Cy5-L-lysine-MESNA and Purification

To N-terminal cysteine Grb2SH2 (N-Cys-Grb2SH2) (200 μM in PBS buffer pH7.2) (200 μl) was added α-D-desthiobiotin-c-Cy5-L-lysine-MESNA (2 mM inreaction buffer) (200 μl). N-Cys-Grb2SH2 was prepared using recombinanttechniques. The reaction buffer consisted of phosphate buffer (200 mM),pH 7.2 containing sodium chloride (200 mM) and 4% MESNA. The reactionmixture was left at RT for 12 hrs, wrapped in foil to protect fromlight. The reaction was then quenched with dl-dithiothreitol (finalconcentration 60 mM). Unreacted dye was separated fromlabelled/unlabelled protein by FPLC, using a fast desalt column andeluent of PBS buffer, pH 7.4; 2 mVmin, detection 280 nm. Proteinfractions were combined and desthiobiotin-e-Cy5-L-lysine affinity probelabelled protein was bound to streptavidin beads (PIERCE Ultralink™streptavidin). The beads were washed vigorously with both PBS buffer andbinding buffer (PBS containing 500 mM NaCl). The product;α-D-desthiobiotin-ε-Cy5-L-Lys-Cys-Grb2SH2 was extracted from thestreptavidin beads by adding cold biotin (1.6 mM). Several extractionruns were required. The materials were further purified by dialysis(PIERCE Slide-a-lyser™ mini dialysis units, 7,000 mwco) to remove freebiotin from the sample. The product was analysed by SDS PAGE togetherwith the following controls (see FIG. 1): Lane 1 MW marker Lane 2Unligated protein control Lane 3 Ligation reaction mixture: α-D-desthiobiotin-ε-Cy5-L-Lysine- MESNA and N-Cys-Grb2SH2 Lane 4Labelled/unlabelled N-Cys-Grb2SH2 after FPLC purification Lane 5 Unboundprotein Lane 6 Streptavidin bead washes Lanes 7, 8, 9 Affinity purifiedproduct Lane 10 Unreacted α-D-desthiobiotin-ε- Cy5-L-Lysine-MESNAThe gel was imaged using a Typhoon imager (FIG. 1B) using parameters forCy5 fluorescence to detect fractions containing the fluorescent label.The gel was then stained with Coomassie blue stain (FIG. 1A) todetermine the protein containing fractions. SDS PAGE gel shows that (a)unlabelled protein (both factor XA and N-Cys-Grb2SH2 did not bind to thestreptavidin beads (FIGS. 1A and 1B, column 5) (enriched protein stain)and (b) the product was removed from the streptavidin beads by addingcold biotin (FIGS. 1A and 1B, columns 7 and 8) (both protein stain andCy5 fluorescence).

1. A compound comprising a cyanine dye or derivative thereof containingat least one target bonding group selected from a carboxylic acidthioester group or a group suitable for covalent reaction with athioester, wherein said compound includes an affinity tag covalentlybound thereto.
 2. The compound of claim 1, having the formula (I):

wherein: D is a dye selected from a cyanine dye or a derivative thereof;B is an affinity tag; F comprises a target bonding group selected from acarboxylic acid thioester group and a 1,2-aminothiol group; M is a groupadapted for attaching to F; and L¹ and L² each independently include agroup containing from 1-40 linked atoms selected from carbon atoms whichmay optionally include one or more groups selected from the groupconsisting of —NR′—, —O—, —CH═CH—, —CO—NH— and phenylenyl groups, whereR′ is selected from hydrogen and C₁-C₄ alkyl.
 3. The compound of claim2, wherein each of L¹ and L² contains from 2 to 30 atoms.
 4. Thecompound of claim 2, wherein L¹ and L² are independently selected fromthe group consisting of:—{(CHR′)_(p)-Q-(CHR′)_(r)}_(s)— where Q is selected from the groupconsisting of: —CHR′—, —NR′—, —O—, —CH═CH—, —Ar— and —CO—NH—; R′ ishydrogen or C₁-C₄ alkyl, p is 0-5, r is 1-5 and s is 1 or
 2. 5. Thecompound of claim 4, wherein Q is selected from the group consisting of—CHR′—, —O— and —CO—NH—, where R′ is hereinbefore defined.
 6. Thecompound of claim 1, wherein said affinity tag is selected from thegroup consisting of biotin and desthiobiotin.
 7. The compound of claim1, wherein said affinity tag is selected from the group consisting ofhis-tag, iminodiacetic acid and nitrilotriacetic acid.
 8. The compoundof claim 2, wherein the target bonding group F is a carboxylic acidthioester of formula:

wherein L′ is a bond or is a group containing from 1-30 linked atomsselected from the group consisting of carbon atoms and carbon atomsincluding one or more groups selected from the group consisting of —NH—,—O— and —CO—NH—; and R″ is C₁-C₄ alkyl, C₆-C₁₀ aryl, or C₇-C₁₅ aralkyl,which may be optionally substituted with sulphonate; or is the group—(CH₂)₂—CONH₂.
 9. The compound of claim 2, wherein the target bondinggroup F is a 1,2-aminothiol group of formula:

wherein L′ is a bond or is a group containing from 1-30 linked atomsselected from the group consisting of carbon atoms and carbon atomsincluding one or more groups selected from the group consisting of —NH—,—O— and —CO—NH—.
 10. The compound of claim 2, having the formula (II):

wherein: groups R³ and R⁴ are attached to the Z¹ ring structure andgroups R⁵ and R⁶ are attached to the Z² ring structure; n is an integerfrom 1 to 3; Z¹ and Z² independently represent the atoms necessary tocomplete one ring or two fused ring aromatic or heteroaromatic systems,each ring having five or six atoms selected from carbon atoms andoptionally no more than two atoms selected from oxygen, nitrogen andsulphur; X and Y are the same or different and are selected from:>CR⁸R⁹, oxygen, sulphur, —CH═CH—, >N—W wherein N is nitrogen and W isselected from hydrogen and the group R¹⁰; at least one of groups R¹, R²,R³, R⁴, R⁵, R⁶, R⁸, R⁹ and R¹⁰ is the group:

groups R⁷ are independently selected from the group consisting ofhydrogen and C₁-C₄ alkyl which may be unsubstituted or substituted witharyl, or two or more of R⁷ together with the group:

form a hydrocarbon ring system substituted with R⁷ and which mayoptionally contain a heteroatom selected from —O—, —S— or >NR⁷;remaining groups R³, R⁴, R⁵ and R⁶ are independently selected from thegroup consisting of hydrogen, halogen, amide, cyano, nitro, mono- ordi-C₁-C₆ alkyl-substituted amino, carbonyl, carboxyl, C₁-C₆ alkyl, C₁-C₆alkoxy, aryl, heteroaryl, aralkyl and the group —(CH₂)_(m)—Y where Y isselected from sulphonate, sulphate, phosphonate, phosphate andquaternary ammonium and m is zero or an integer from 1 to 6; remaininggroups R⁸, R⁹ and R¹⁰ are independently C₁-C₆ alkyl; and remaininggroups R¹ and R² are independently selected from hydrogen, C₁-C₁₀ alkyl,the group —(CH₂)_(m)—Y wherein Y and m are hereinbefore defined, andbenzyl which may be unsubstituted or substituted by up to two nitrogroups.
 11. The compound of claim 2 having the formula (III):

wherein groups R¹², R¹³, R¹⁴ and R¹⁵ are attached to the ringscontaining X and Y or, optionally are attached to atoms of the Z¹ and Z²ring structures; Z¹ and Z² independently represent the atoms necessaryto complete one ring or two fused ring aromatic or heteroaromaticsystems, each ring having five or six atoms selected from carbon atomsand optionally no more than two atoms selected from oxygen, nitrogen andsulphur; X and Y are the same or different and are selected from:>CR⁸R⁹, oxygen, sulphur, —CH═CH—, >N—W wherein N is nitrogen and W isselected from hydrogen and the group R¹⁰; A is selected from O and NR¹⁶where R¹⁶ is the substituted amino radical:

at least one of groups R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁷ and R¹⁸is the group:

remaining groups R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ are independently selectedfrom the group consisting of hydrogen, halogen, amide, cyano, nitro,mono- or di-C₁-C₆ alkyl-substituted amino, carbonyl, carboxyl, C₁-C₆alkyl, C₁-C₆ alkoxy, aryl, heteroaryl, aralkyl and the group—(CH₂)_(m)—Y where Y is selected from sulphonate, sulphate, phosphonate,phosphate and quaternary ammonium and m is zero or an integer from 1 to6; remaining groups R⁸, R⁹ and R¹⁰ are independently C₁-C₆ alkyl;remaining group R¹⁷ is selected from hydrogen, C₁-C₄ alkyl and aryl; andremaining group R¹⁸ is selected from C₁-C₆ alkyl, aryl, heteroaryl, anacyl radical having from 2-7 carbon atoms, and a thiocarbamoyl radical.12. The compound of claim 10, wherein Z¹ and Z² are selectedindependently from the group consisting of phenyl, pyridinyl, naphthyl,quinolinyl and indolyl moieties.
 13. The compound of claim 110 whereinZ¹ and Z² are selected from phenyl and naphthyl moieties.
 14. A methodfor labelling a protein of interest wherein said protein contains or isderivatised to contain an N-terminal cysteine, the method comprising: i)adding to a liquid containing said protein a compound of formula (I):

wherein: D is a dye selected from a cyanine dye or a derivative thereof;B is a bioaffinity tag; F comprises a target bonding group selected froma carboxylic acid thioester group and a 1,2-aminothiol group; M is agroup adapted for attaching to F; and L¹ and L² each independentlyinclude a group containing from 1-40 linked atoms selected from carbonatoms which may optionally include one or more groups selected from—NR′—, —O—, —CH═CH—, —CO—NH— and phenylenyl groups, where R′ is selectedfrom hydrogen and C₁-C₄ alkyl; and ii) incubating said compound withsaid protein under conditions suitable for labelling said protein. 15.The method of claim 14, wherein each of L¹ and L² contains from 2 to 30atoms.
 16. The method of claim 14, wherein L¹ and L² are independentlyselected from the group:—{(CHR′)_(p)-Q-(CHR′)_(r)}_(s)— where Q is selected from the groupconsisting of: —CHR′—, —NR′—, —O—, —CH═CH—, —Ar— and —CO—NH—; R′ ishydrogen or C₁-C₄ alkyl, p is 0-5, r is 1-5 and s is 1 or
 2. 17. Themethod of claim 16, wherein Q is selected from the group consisting of—CHR′—, —O— and —CO—NH—, where R′ is hereinbefore defined.
 18. Themethod of claim 14, further comprising separating and/or purifying thedye-labelled protein of interest by affinity chromatography.
 19. Themethod of claim 14, wherein said protein of interest is selected fromantibody, the group consisting of antibodies, antigens, proteinspeptides, microbial materials, cells and cell membranes.
 20. Thecompound of claim 11, wherein Z¹ and Z² are selected independently fromthe group consisting of phenyl, pyridinyl, naphthyl, quinolinyl andindolyl moieties.
 21. The compound of claim 11, wherein Z¹ and Z² areselected from phenyl and naphthyl moieties.